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Mge1 Functions As a Nucleotide Release Factor for Ssc1, a Mitochondrial Hsp70 of Saccharomyces Cerevisiae

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Mge1 Functions As a Nucleotide Release Factor for Ssc1, a Mitochondrial Hsp70 of Saccharomyces Cerevisiae JMB MS 1679 [15/1/97] J. Mol. Biol. (1997) 265, 541±552 Mge1 Functions as a Nucleotide Release Factor for Ssc1, a Mitochondrial Hsp70 of Saccharomyces cerevisiae Bingjie Miao, Julie E. Davis and Elizabeth A. Craig* Department of Biomolecular Mge1, a GrpE-related protein in the mitochondrial matrix of the budding Chemistry, 1300 University yeast Saccharomyces cerevisiae, is required for translocation of precursor Avenue, University of proteins into mitochondria. The effect of Mge1 on nucleotide release from Wisconsin, Madison, WI 53706 Ssc1, an Hsp70 of the mitochondrial matrix, was analyzed. The release of USA both ATP and ADP from Ssc1 was stimulated in the presence of Mge1, therefore we conclude that Mge1 functions as a nucleotide release factor for Ssc1. Mge1 bound stably to Ssc1 in vitro; this interaction was resistant to high concentrations of salt but was disrupted by the addition of ATP. ADP was much less effective in releasing Mge1 from Ssc1 whereas ATPgS and AMPPNP could not disrupt the Ssc1/Mge1 complex. Ssc1-3, a temperature sensitive SSC1 mutant protein, did not form a detectable complex with Mge1. Consistent with the lack of a detectable interaction, Mge1 did not stimulate nucleotide release from Ssc1-3. A conserved loop structure on the surface of the ATPase domain of DnaK has been impli- cated in its interaction with GrpE. Since the single amino acid change in Ssc1-3 lies very close to the analogous loop in Ssc1, the role of this loop in the Ssc1:Mge1 interaction was investigated. Deletion of the loop abol- ished the physical and functional interaction of Ssc1 with Mge1, suggesting that the loop in Ssc1 is also important for the Ssc1:Mge1 inter- action. Two mutants with single amino acid changes within the loop did not eliminate the stable binding of Mge1, yet the binding of Mge1 did not stimulate the release of nucleotides from the mutant SSC1 proteins. We propose that the loop region of Ssc1 is important for the physical interaction between Mge1 and Ssc1, and for generation of a confor- mational change necessary for Mge1-induced nucleotide release. # 1997 Academic Press Limited *Corresponding author Keywords: Hsp70; Mge1; Ssc1; nucleotide release; chaperone Introduction terminal ATPase domain, which binds and hydro- lyzes ATP, and a somewhat less conserved pep- The 70-kDa heat shock proteins (Hsp70s) have tide binding domain (Chappell et al., 1987; Wang been highly conserved during evolution and are et al., 1993; Freeman et al., 1995). The tertiary struc- present in every organism examined so far. ture of the 44-kDa ATPase domain is similar to Hsp70s are important for a variety of cellular func- that of hexokinase and actin (Flaherty et al., 1990, tions, including protein folding, protein transloca- 1991; Bork et al., 1992), whereas the C-terminal tion across biological membranes, and protein peptide binding domain has a unique b-sandwich degradation (for reviews, see Craig et al., 1993; structure followed by an extended structure of a- Hartl, 1996; Morimoto et al., 1994). Hsp70s have helices (Zhu et al., 1996). The interaction between two functional domains, a highly conserved N- the two domains is critical for the function of Hsp70s (Buchberger et al., 1994b, 1995). Hsp70s function as molecular chaperones by binding to Abbreviations used: ATPgS, adenosine 50-O-(3- short stretches of hydrophobic peptide sequences thiotriphosphate); AMPPNP, 50-adenylyl-b,g- imidodiphosphate; 5-FOA, 5-¯uoroorotic acid; GST, thus preventing premature folding or aggregation glutathione S-transferase; IPTG, isopropyl b-D- of partially unfolded proteins (Flynn et al., 1989; thiogalactopyranoside; PEI-cellulose, polyethyleneimine- Blond-Elguindi et al., 1993; Gragerov et al., 1994). cellulose; TLC, thin layer chromatography. Upon ATP binding and/or hydrolysis, bound 0022±2836/97/050541±12 $25.00/0/mb960762 # 1997 Academic Press Limited JMB MS 1679 [15/1/97] 542 Mge1, a Nucleotide Release Factor for Ssc1 peptide is released to allow for its proper folding Gambill et al., 1993). Ssc1 has been found to be as- (Palleros et al., 1993; McCarty et al., 1995; Banecki sociated with precursor proteins during and after & Zylicz, 1996). This cycle of ATP binding and translocation (Ostermann et al., 1990; Scherer et al., hydrolysis coupled to peptide binding and release 1990; Manning-Krieg et al., 1991), consistent with is essential for the function of Hsp70s. its role in the import and maturation of precursor Several lines of evidence indicate that DnaK, an proteins. The binding of Ssc1 to precursor proteins Hsp70 protein of Escherichia coli, functions together in transit across the mitochondrial membranes is with two proteins, DnaJ and GrpE (reviewed by essential for conferring the unidirectionality of the Georgopoulos et al., 1994). Mutations in the dnaK, import process (Ungermann et al., 1994). Mge1 can dnaJ or grpE genes result in similar phenotypes; be quantitatively co-immunoprecipitated with Ssc1 furthermore, DnaK, DnaJ and GrpE function from isolated mitochondria, and both Ssc1 and together in a variety of in vitro assays, such as in- Mge1 can be co-immunoprecipitated with a pre- itiation of l DNA replication and refolding of de- cursor protein that is trapped at the import site natured proteins. DnaJ and GrpE exert their (Voos et al., 1994), suggesting a functional co- effects, at least in part, by modulating the ATPase operation between Ssc1 and Mge1 in the process activity of DnaK. DnaJ stimulates the hydrolysis of of protein translocation. bound ATP by DnaK, whereas GrpE promotes the Because of the sequence similarity between Mge1 release of nucleotides from DnaK (Liberek et al., and GrpE, and the functional cooperation between 1991a). DnaJ alone stimulates the steady-state Ssc1 and Mge1, it has been hypothesized that ATPase activity of DnaK by two to tenfold, Mge1 functions as a nucleotide release factor for whereas GrpE alone has a minimal effect (Jordan Ssc1. Here we show that Mge1 is in fact a & McMacken, 1995; McCarty et al., 1995). How- nucleotide release factor for Ssc1. We also charac- ever, DnaJ and GrpE together can stimulate the terized the Ssc1:Mge1 interaction and examined steady-state ATPase activity of DnaK by up to the effect of mutations of the loop in Ssc1, which 100-fold. is analogous to the GrpE-interacting loop in DnaK, Unlike the DnaK:DnaJ interaction, GrpE binds on the interaction of Ssc1 with Mge1. Based on tightly to DnaK. The complex between DnaK and these results, a possible mechanism for Mge1- GrpE is stable in the presence of high concen- induced nucleotide release is discussed. trations of salt, but is disrupted upon the addition of ATP (Zylicz et al., 1987). GrpE binds to the 44- Results kDa ATPase domain of DnaK, and a conserved loop structure on the surface of the ATPase Mge1 is a nucleotide release factor for Ssc1 domain has been implicated in the interaction of DnaK with GrpE (Buchberger et al., 1994a). A Since GrpE functions as a nucleotide release factor point mutation in this loop as well as a deletion of for DnaK, we wanted to test whether Mge1 is this loop eliminates the physical and functional able to function as a nucleotide release factor for interaction between DnaK and GrpE. It has been Ssc1. Consistent with the lack of an effect of GrpE proposed that the binding of GrpE induces a con- on the ATPase activity of DnaK (Jordan & formational change in DnaK, thus triggering McMacken, 1995), Mge1 had a minimal effect on nucleotide release (Buchberger et al., 1994a), yet the ATPase activity of Ssc1 (data not shown). To the mechanism of GrpE-induced nucleotide release examine more closely the interaction of Mge1 with remains to be elucidated. Ssc1, an isolated step in the ATPase reaction, the Numerous eukaryotic Hsp70s and several DnaJ- ATP hydrolysis step, was analyzed by single turn- related proteins have been identi®ed in eukaryotic over experiments. Complexes of Ssc1 and 32 cells. Genetic and biochemical studies have estab- [a- P]ATP were formed at 30C; isolation of the lished the functional interaction between them complex by size exclusion chromatography was (reviewed by Cyr et al., 1994). Mge1 (also referred carried out at 4C to limit the hydrolysis of to as Yge1p, GrpEp), a GrpE-related protein, has [a-32P]ATP. The isolated Ssc1/ATP complex was recently been identi®ed in the mitochondrial then incubated at 30C and the hydrolysis of matrix of Saccharomyces cerevisiae (Laloraya et al., bound ATP monitored. As shown in Figure 1A, 1994; Bolliger et al., 1994; Ikeda et al., 1994). Mge1, wild-type Ssc1 hydrolyzed bound ATP with about which shares 34% identity with Escherichia coli 50% of the bound ATP being hydrolyzed within GrpE (Laloraya et al., 1994), is essential for the four minutes of incubation. To investigate the growth of S. cerevisiae. Mge1 is required for nor- stability of the interaction of ATP with Ssc1, the mal import and maturation of nuclear-encoded ability of excess unlabeled ATP or ADP to quench mitochondrial proteins (Laloraya et al., 1994, 1995; the hydrolysis of the bound [a-32P]ATP was deter- Westerman et al., 1995). mined. Assuming that there is no signi®cant coop- Ssc1, an essential Hsp70 in the mitochondrial erativity in nucleotide binding to Ssc1, addition of matrix, is also required for the translocation and unlabeled nucleotide should have no effect on the maturation of precursor proteins, as temperature- hydrolysis of ATP which remains bound to Ssc1. sensitive mutations in SSC1 result in a block of However, [a-32P]ATP which is released prior to import of precursor proteins upon shifting to the hydrolysis must then compete with the vast excess non-permissive temperature (Kang et al., 1990; of unlabeled nucleotide for rebinding before JMB MS 1679 [15/1/97] Mge1, a Nucleotide Release Factor for Ssc1 543 Figure 1.
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